Single nucleotide polymorphism detection by optical DNA-based sensing coupled with whole genomic amplification.

The work presented here deals with the optimization of a strategy for detection of single nucleotide polymorphisms based on surface plasmon resonance imaging. First, a sandwich-like assay was designed, and oligonucleotide sequences were computationally selected in order to study optimized conditions for the detection of the rs1045642 single nucleotide polymorphism in the gene ABCB1. Then the strategy was optimized on a surface plasmon resonance imaging biosensor using synthetic DNA sequences in order to evaluate the best conditions for the detection of a single mismatching base. Finally, the assay was tested on DNA extracted from human blood which was subsequently amplified using a whole genome amplification kit. The direct detection of the polymorphism was successfully achieved. The biochip was highly regenerable and reusable for up to 20 measurements. Furthermore, coupling these promising results with the multiarray assay, we can foresee applying this biosensor in clinical research extended to concurrent analysis of different polymorphisms.

Direct detection of genomic DNA by surface plasmon resonance imaging: an optimized approach.

The direct detection of specific sequences in genomic DNA samples is very challenging in the biosensor-based approach. In this work we developed an optimized strategy for the direct detection of DNA sequences in human genomic samples by a surface plasmon resonance imaging technology. As model study, the target analyte was identified in a DNA sequence mapping the human ABCB1 gene. The computed-assisted approach was here applied for probe design. After a preliminary evaluation of the probe functioning by the complementary synthetic target, the system was applied to the direct detection of the target sequence in human genomic DNA extracted from lymphocytes. To achieve this result, several steps aimed to improve the analytical performances of the biosensor were studied and optimized. The immobilization chemistry, based on thiolated probes, was adapted here to non-amplified sequence detection. DNA sample pre-treatments, i.e. genomic fragmentation by ultrasounds and dsDNA denaturation by thermal treatment were also investigated. A sandwich-like strategy, by using a secondary probe, was also applied to understand and confirm the selectivity of the developed biosensor in detecting ABCB1 gene in genomic samples. Finally, a reliable calibration curve of ABCB1 was obtained with an experimental detection limit of 140 aM. Furthermore, the biosensor was well regenerable, assuring up to thirty cycles of effective measurements.

SPR detection of human hepcidin-25: a critical approach by immuno- and biomimetic-based biosensing.

The human hepcidin-25 hormone has a key role in iron regulation in blood. The clinical relevance of this hepatic ~2.8 kDa cysteine-rich peptide is rapidly increasing, since altered levels can be associated with inflammatory events and iron dysfunctions, such as hereditary hemochromatosis and iron overload. Moreover, hepcidin has also attracted the anti-doping field for its possible role as indirect marker of erythropoietin blood doping. Methods currently reported are based on immunoassays (ELISA and RIA), or various types of mass spectroscopy (MS)-based protocols, semi-quantitative or quantitative. Despite the great effort in optimizing robust and simple assays measuring hepcidin in real matrices, at present this challenge remains still an open issue. To explore the possibility to face hepcidin detection through the development of affinity-based biosensors, we set up a comparative study by surface plasmon resonance (SPR) technology. An immuno-based, on anti-hepcidin-25 IgG, and a biomimetic-based, on a synthetic peptide corresponding to the hepcidin-binding site on ferroportin (HBD), biosensors were developed. Here we report behaviors and analytical performances of the two systems, discussing limits and potentialities.

A rational approach in probe design for nucleic acid-based biosensing.

Development of nucleic acid-based sensing attracts the interest of many researchers in the field of both basic and applied research in chemistry. Major factors for the fabrication of a successful nucleic acid sensor include the design of probes for target sequence hybridization and their immobilization on the chip surface. Here we demonstrate that a rational choice of bioprobes has important impact on the sensor's analytical performances. Computational evaluations, by a simple and freely available program, successfully led to the design of the best probes for a given target, with direct application to nucleic acid-based sensing. We developed here an optimized and reproducible strategy for in silico probe design supported by optical transduction experiments. In particular Surface Plasmon Resonance imaging (SPRi), at the forefront of optical sensing, was used here as proof of principle. Five probes were selected, immobilized on gold chip surfaces by widely consolidated thiol chemistry and tested to validate the computational model. Using SPRi as the transducting component, real-time and label free analysis was performed, taking the Homo sapiens actin beta (ACTB) gene fragment as model system in nucleic acid detection. The experimental sensor behavior was further studied by evaluating the strength of the secondary structure of probes using melting experiments. Dedicated software was also used to evaluate probes' folding, to support our criteria. The SPRi experimental results fully validate the computational evaluations, revealing this approach highly promising as a useful tool to design biosensor probes with optimized performances.

Prevalence of celiac disease in patients with juvenile chronic arthritis.

We estimated the prevalence of celiac disease in children with juvenile chronic arthritis (JCA), using antiendomysium antibodies as the screening test to select patients for intestinal biopsy. We studied 119 children with JCA and found four patients with antiendomysium antibodies. In three of these patients (2.5%), intestinal biopsy revealed villous atrophy; in the fourth the intestinal mucosa was normal. We conclude that the prevalence of celiac disease is increased in patients with JCA.